Toolkits for GEaReD
For the succesfull implementation of GEaReD, technological improvements at several stages are needed. 1) Efficient phenotyping technologies are required. Wild plants can be more phenotypically diverse and they are not well described in terms of agronomic traits compared to domesticated crops. It is crucial that the right representatives with the desirable traits within the species are selected for GEaReD. 2) DNA sequence information of wild plants is also often sparse. Genome sequencing efforts of candidate species such as wild relatives of current crops is a prerequisite for releasing the full potential of GEaReD. The combination of these improvements will lead to a further increase in data and requires new data processing solutions. Artificial Intelligence will be a key technology for this, and first generation AI is already employed for genomic data processing. In the future, AI will further support scientist with acquiring and connecting phenotyping data with omic data enabling the construction of large databases. 3) Transformation methods, usually involving tissue culture, are needed to facilitate genome editing in candidate species. Even in many of our crops, tissue culture technologies are limited to be efficient in only a few cultivars of the crop. Ideally, there should be no such constraints in a candidate species for GEaReD. 4) A platform of molecular tools for precision genome editing over a wide range of species is essential for releasing the full potential of the genome editing technologies. Among these, the most frequently used tool is the CRISPR/Cas9 mediated genome editing. However, recent progress has already provided many new methodologies for targeted mutagenesis in the plant genome. First, concurrent mutagenesis of multiple genes were made possible through multiplex genome editing. This development was further accentuated through the development of alternatives to the canonical PAM site (NCC). Secondly, the function of the endonuclease were modulated to provide a nickase activity that creates single strand breaks and allow site specific genomic integration [10]. Base editing of specific nucleotides is one of the latest developments. By introducing deaminases or so-called base-editors, together with for example a nickase, specific C-G base pairs can be changed to T-A base pairs and vice versa [11]. In another technology, the fusion of a transposase/recombinase to hijack a transposon system, enables the introduction of sequences at a predetermined location [11]. A similar approach to these transposon systems, but more elegant way is prime-CRISPR. Hereby a reverse transcriptase is fused to an endonuclease, enabling to integrate small sequences at specific target sites in the genome [11]. These two new techniques are perfectly suited to alter the activity of promoters, but could also be used to re-design proteins. Changes in the amino acid sequence could be used to alter activity, phosphorylation or localization of proteins.